Effects of 1,10-phenanthroline on metalloproteinase activity and the inhibition of <i>T. vaginalis</i>-induced apoptosis.

(A) Micrographs of SiHa cells incubated with live T. vaginalis or with metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT, 5 mM) pretreated T. vaginalis for 16 h. (B) SiHa cells were treated with live T. vaginalis or incubated with 1,10-PT for 16 h and then the protein extracts were analyzed by western blotting using indicated antibodies. (C) SiHa cells incubated with live T. vaginalis (MOI = 2) or, 1,10-PT-pretreated T. vaginalis for 16 h. The cells were collected, and DNA fragmentation was determined by agarose-gel electrophoresis. (D) Live T. vaginalis, T. vaginalis ESP and 1,10-PT-pretreated T. vaginalis ESP were subjected to electrophoresis. Substrate proteolytic activity was determined by 10% SDS-PAGE with 0.1% gelatin. (E) SiHa cells were treated as in Fig. 2C, the supernatants were collected and then the protease activities were determined by casein-hydrolysis assay. Each assay was carried out in triplicate, and the results shown are the relative percentages of protease activities in the cells infected with the pretreated parasites compared with those in the T. vaginalis-infected cells, which were set as 100%. * P<0.05. (F) The cytosolic fraction (only for dectection of cytochrome c) or protein extracts (detection for the others beside of cytochrome c) of the SiHa cells were isolated, and western blotting was performed with using indicated antibodies. A representative result of three independent replicates is shown.